The invention relates to methods of preventing the transformation of normal mammalian cells into tumor cells.
Huge amounts of time and money have been spent to better understand cancer and searching for ways to prevent and cure cancer. The results of these research efforts have provided a greater understanding of the biological and biochemical events that participate in the formation of tumors.
Tumor cells display a variety of characteristics that distinguish them from normal cells. Recent studies in the molecular genetics of cancer indicate that certain genes known as oncogenes may play a role in the transformation of some cells from their normal condition to a cancerous condition.
An oncogene which encodes a protein that exposes antigenic sites on the surface of transformed cells has been identified by transfection of DNA from ethyl nitrosourea-induced rat neuroblastomas into NIH3T3 cells. This oncogene has been termed neu. The neu gene has been found to be amplified in some human tumors, particularly those of the breast, suggesting that this gene may play a role in the etiology of human cancer.
The neu oncogene encodes a cell surface protein on rat cells transformed by it. The protein encoded by the neu oncogene is a 185 kDa transmembrane glycoprotein with tyrosine kinase activity, generally known by the name p185. The neu gene is closely related to the epidermal growth factor (EGF) receptor gene in structure.
The neu oncogene and p185 have also been found active in human adenocarcinomas including breast, lung, salivary gland and kidney adenocarcinomas, as well as prostate neuroblastoma. In human primary breast cancers, amplification of the neu oncogene was found in about 30% of all malignant tumors examined. Increased stage of malignancy, characterized by large tumor size and increased number of positive lymph nodes as well as reduced survival time and decreased time to relapse, was directly correlated with an increased level of amplification of the neu gene. The neu protooncogene is expressed at low levels in normal human tissues. Further, neu has been associated with 100% of the ductal carcinomas studied in situ, Lodato, R. F., et al. (1990) Modern Pathol. 3(4):449.
While changes in diet and behavior can reduce the likelihood of developing cancer, it has been found that some individuals have a higher risk of developing cancer than others. Further, those individuals who have already developed cancer and who have been effectively treated face a risk of relapse and recurrence.
Advancements in the understanding of genetics and developments in technology as well as epidemiology allow for the determination of probability and risk assessment an individual has for developing cancer. Using family health histories and/or genetic screening, it is possible to estimate the probability that a particular individual has for developing certain types of cancer. Those individuals that have been identified as being predisposed to developing a particular form of cancer can take only limited prophylactic steps towards reducing the risk of cancer. There is no currently available method or composition which can chemically intervene with the development of cancer and reduce the probability a high risk individual will develop cancer.
Similarly, those individuals who have already developed cancer and who have been treated to remove the cancer or are otherwise in remission are particularly susceptible to relapse and reoccurrence.
There is a need for improved preventative agents for individual with a high risk to develop cancer and for individuals who have had cancer enter remission or be removed. In cases where the type of cancer the individual is at risk to develop, such as tumors associated with neu, there is a need for specific agents which can be administered to reduce the probability that a predisposed individual will develop cancer or that a patient in remission will suffer a relapse.
The present invention provides methods for the prevention of tumor cells which express a translation product of the neu oncogene on their surfaces. In accordance with the invention, a prophylactic amount of an antibody that specifically binds to p185 is administered to an individual.
The present invention provides methods of preventing the transformation of normal human cells into tumors cells which express a translation product of the neu oncogene on their surfaces. In accordance with the invention, a prophylactic amount of an antibody that specifically binds to p185 is administered to an individual.
The present invention provides methods for the prevention of the origination of genetically induced mammalian tumor cells which express a translation product of the neu oncogene on their surfaces by interfering with a transforming event. In accordance with the invention, a prophylactic amount of an antibody that specifically binds to p185 is administered to an individual.
As used herein, the terms xe2x80x9cneu-associated cancerxe2x80x9d and xe2x80x9cneu-associated tumorsxe2x80x9d are meant to refer to tumor cells and neoplasms which express the neu gene to produce p185.
The translation product of the neu oncogene is p185, a transmembrane glycoprotein having tyrosine kinase activity and a molecular weight of about 185,000 daltons as determined by carrying out electrophoresis on the glycoprotein and comparing its movement with marker proteins of known molecular weight. Experiments have shown that administration of an antibody binding to p185 results in the reduced incidence of neu-associated tumors in a population susceptible to such tumors. Anti-p185 antibodies selectively inhibit the neoplastic development in animals susceptible to developing neu transformed tumors.
The occurrence of mammalian tumors cells which express a translation product of the neu oncogene on their surfaces can be prevented by administration of antibodies which bind to p185. In accordance with the invention, a prophylactic amount of an antibody that specifically binds to p185 is administered to an individual who is identified as being susceptibleto neu-associated tumors.
The present invention is particularly useful to prophylactically treat an individual who is predisposed to develop neu-associated tumors or who has had neu-associated tumors and is therefore susceptible to a relapse or recurrence.
As used herein, the term xe2x80x9chigh risk individualxe2x80x9d is meant to refer to an individual who has had a neu-associated tumor either removed or enter remission and who is therefore susceptible to a relapse or recurrence. As part of a treatment regimen for a high risk individual, the individual can be prophylactically treated against the neu-associated tumors that they have been diagnosed as having had in order to combat a recurrence. Thus, once it is known that an individual has had cancer characterized by tumor cells with p185 on their cell surfaces, the individual can be treated according to the present invention to prevent normal cells from transforming into tumor cells.
Prophylactic compositions for prevention of neu-associated tumors comprise an antibody specific for the p185 molecule and a pharmaceutically acceptable carrier. According to preferred embodiments, the prophylactic compositions for prevention of neu-associated tumors are injectable. The compositions comprise an antibody specific for the p185 molecule and a pharmaceutically acceptable carrier or injection vehicle.
The antibodies are chosen from antibodies made according to the procedures described in detail below or other conventional methods for producing monoclonal antibodies. The carrier be selected from those well known to persons having ordinary skill in the art. An example of a carrier is sterile saline.
Antibodies Specific for Rat and Human p185
Those having ordinary skill in the art can produce monoclonal antibodies which specifically bind to p185 and are useful in prophylactic anti-tumor compositions using standard techniques and readily available starting materials. The techniques for producing monoclonal antibodies are outlined in Harlow, E. and D. Lane, (1988) ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., which is incorporated herein by reference, provide detailed guidance for the production of hybridomas and monoclonal antibodies which specifically bind to target proteins.
Briefly, the protein of interest, rodent or human p185 for example, is injected into mice. The spleen of the mouse is removed, the spleen cells are isolated and fused with immortalized mouse cells. The hybrid cells, or hybridomas, are cultured and those cells which secrete antibodies are selected. The antibodies are analyzed and, if found to specifically bind to the protein of interest, the hybridoma which produces them is cultured to produce a continuous supply of antigen specific antibodies.
According to the present invention, antibodies specific for either rodent, particularly rat, p185 or the corresponding human p185 may be used in prophylactic compositions. Accordingly, either rodent p185 or human p185 is used to generate hybridomas. In both cases, the genes which encode these proteins are widely known and readily available to those having ordinary skill in the art. Thus, one having ordinary skill in the art can make antibodies useful to practice the present invention. In addition to rodent antibodies, the present invention relates to human antibodies, humanized antibodies, Fabs and chimeric antibodies and Fabs which bind to p185 which may be produced routinely by those having ordinary skill in the art.
In some preferred embodiments of the present invention, the prophylactic composition comprises monoclonal antibodies designated 7.5.5, 7.9.5, 7.16.4 and 7.21.2. In some preferred embodiments of the present invention, the prophylactic composition comprises humanized monoclonal antibodies or Fabs which contain complementarity determining regions from antibodies designated 7.5.5, 7.9.5, 7.16.4 and 7.21.2. In some preferred embodiments of the present invention, the prophylactic composition comprises humanized monoclonal antibodies or Fabs which contain variable regions from antibodies designated 7.5.5, 7.9.5, 7.16.4 and 7.21.2.
Patient Population
Although the present invention may be used to prevent tumors in any patient population identified as being susceptible to neu-associated tumors, it is particularly useful in high risk individuals who, for example, have a family history of neu-associated cancer or show a genetic predisposition. Additionally, the present invention is particularly useful to prevent neu-associated tumors in patients who have had neu-associated tumors removed by surgical resection or who have been diagnosed as having neu-associated cancer in remission.
Those having ordinary skill in the art can readily identify individuals who are susceptible to neu-associated tumors, particularly those individuals considered to be a high risk for whom the methods of the invention are particularly useful.
Compositions
The prophylactic compositions may include additional components to render them more effective. For example, a prophylactic composition of the invention may comprise multiple anti-p185 antibodies including antibodies specific for different epitopes of p185.
The prophylactic compositions may include other anti-cancer agents such as, for example, cis-platin. As a step in the method of the invention, chemotherapeutics may be administered prophylactically to patients who have treated for neu-associated cancer by surgery or radiation treatment and who have had removal or remission.
Administration Regimen
About 5 xcexcg to 5000 mg of antibody may be administered. In some preferred embodiments, 50 xcexcg to 500 mg of antibody may be administered. IN other preferred embodiments, 500 xcexcg to 50 mg of antibody may be administered. In a preferred embodiment, 5 mg of antibody is administered.
Prophylactic compositions may be administered by an appropriate route such as, for example, by oral, intranasal, intramuscular, intraperitoneal or subcutaneous administration. In some embodiments, intravenous administration is preferred.
Subsequent to initial administration, individuals may be boosted by readministration. In some preferred embodiments, multiple administrations are performed.